Loss of Myo19 increases metastasis by enhancing microenvironmental ROS gradient and chemotaxis

Tumor metastasis involves cells migrating directionally in response to external chemical signals. Reactive oxygen species (ROS) in the form of H2O2 has been demonstrated as a chemoattractant for neutrophils but its spatial characteristics in tumor microenvironment and potential role in tumor cell dissemination remain unknown. Here we investigate the spatial ROS distribution in 3D tumor spheroids and identify a ROS concentration gradient in spheroid periphery, which projects into a H2O2 gradient in tumor microenvironment. We further reveal the role of H2O2 gradient to induce chemotaxis of tumor cells by activating Src and subsequently inhibiting RhoA. Finally, we observe that the absence of mitochondria cristae remodeling proteins including the mitochondria-localized actin motor Myosin 19 (Myo19) enhances ROS gradient and promotes tumor dissemination. Myo19 downregulation is seen in many tumors, and Myo19 expression is negatively associated with tumor metastasis in vivo. Together, our study reveals the chemoattractant role of tumor microenvironmental ROS and implies the potential impact of mitochondria cristae disorganization on tumor invasion and metastasis.

16th Jun 2023 1st Editorial Decision Dear Prof. Wu, Thank you for submitting your manuscript to EMBO Reports.I have read your study carefully and discussed it with the other members of our editorial team including our chief editor Dr. Bernd Pulverer.I regret to inform you that we have decided not to pursue publication of this manuscript in its current form, but we would be happy to reconsider it with some additional analysis as mentioned below.
I apologize for this unusual delay in getting back to you, which was caused by the current high rate of new submissions to our office, affecting our usually much shorter editorial handling time.
We appreciate your findings supporting that loss of Myo19 in cancer enhances the H2O2 gradient, thereby promoting invasive behavior of cancer cells.We realize that these findings are as such of interest to the field.However, we also note that, in our view, whether the changes in H2O2 gradient is necessary for the Myo19 depletion induced spheroid invasion has not been conclusively demonstrated -e.g. by comparing spheroid invasion of WT and KO spheroids in the presence of catalase.We feel that these points would come up during peer-review as well.As such, we concluded that the advance provided is not sufficient for publication in EMBO Reports in the current form of the manuscript.That said, we would be happy to send the manuscript out for formal peer-review should you be willing to include additional data addressing these concerns.
Thank you for giving us the opportunity to consider this manuscript.
Yours sincerely, Deniz Senyilmaz Tiebe, PhD Scientific Editor EMBO Reports ** As a service to authors, EMBO Press provides authors with the ability to transfer a manuscript that one journal cannot offer to publish to another journal, without the author having to upload the manuscript data again.To transfer your manuscript to another EMBO Press journal using this service, please click on Link Not Available June 23, 2023   The editorial team

EMBO Reports
To the Editor, Please find in the attachment our re-submission entitled "Loss of Myo19 increases metastasis by enhancing microenvironmental ROS gradient and chemotaxis".We thank the editor and the editorial team for the valuable suggestions.We have added additional experiments accordingly.We found that spheroid invasion was enhanced in Myo19 knock out (KO) spheroids with sharper ROS gradient, and treatment of catalase could inhibit the invasiveness in both wild type (WT) and Myo19 KO spheroids (Fig. 3C, and line 154-155), arguing that enhanced microenvironmental ROS gradient in Myo19depleted spheroids resulted in their increased invasiveness.
We feel that the additional data have significantly strengthened the conclusions of this manuscript and would like to re-submit it for potential peer review.We deeply appreciate your efforts in handling our manuscript.Thank you for the submission of your research manuscript to our journal, which was now seen by three referees, whose reports are copied below.
We concur with the referees that the proposed role of microenvironmental ROS gradient in tumor cell chemotaxis mediated by Src kinase is in principle interesting.However, the referees also raise significant concerns that need to be addressed to consider publication here.
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I look forward to seeing a revised version of your manuscript when it is ready.Please let me know if you have questions or comments regarding the revision.This work represents the natural continuation of a recent investigation published by authors in Nature Communication (Mechanical instability generated by Myo19 contributes to mitochondria cristae architecture and OXPHOS" and PMID: 35562374), in which they describe the relationship between Myo19 and mitochondrial metabolism.Here, the authors want to demonstrate that these effects mediated by Myo19 control tumor invasion and metastasis, particularly by regulating the tumor microenvironmental ROS.This new study is fascinating and may extend the knowledge of the important role of ROS microenvironment in tumor growth and, especially, in metastasis.
However, I would ask the authors to perform some experiments to strengthen their data.One concern is regarding the in vivo experiments reported in Figure 1.The authors injected mice with 4T1 scramble, Mic60 KD, and Myo19 KD cells.As reported in the text, they affirmed that myo19 KO led to higher invasiveness (Fig 1E).However, in the graph, it is the Mic60 KD that gives the higher rate of invasiveness.Myo19KD has a low significance of p=0.0405.This also occurs in Fig. 1F.Have the authors inverted the graphs?If the label is correct, authors should explain the choice to focus their investigation on myo19.Throughout the text, the authors tried to justify this only at line 118, but this is a weak connection that further experiments should validate.Furthermore, why did the authors not investigate the contribution of Mic60 throughout the manuscript?If Mic60 is important for ROS production and mitochondrial modeling, why did the authors not measure its levels in human samples?Another thing that is not clear is that even if they found significant increases in the invasiveness, the authors did not find variations in the tumor growth.This is very strange, and the authors should justify this.To be sure about all these in vivo results, I encourage authors to perform a metastatic mouse model in which the primary tumor is removed.Moreover, can the metastatic potential reduce if mice are injected with cells overexpressing myo19?In line with this, using a 3D system is a good model to study invasiveness.However, they are "produced" by immortalized cells.Can some key experiments be conducted using metastatic cells obtained from in vivo experiments?Moreover, the mitochondrial distribution, the levels of key proteins of the study (such as Src), the metabolism, the ROS abundance, and the levels of ROS scavenger factors (only to cite a few) should also be detected in samples obtained from in vivo experiments.This will greatly improve the quality of the investigation.
Another critical point is that the authors investigate mitochondrial dynamics and metabolism.I agree with them that their previous work deeply investigates the relationship between mitochondria and myo19.However, they should verify the activity and the dynamics of mitochondria in the new experimental conditions reported in this work (experiments reported in S3 are insufficient to support this).
Similarly, authors should further validate the importance of ROS by increasing and decreasing their levels.The authors performed experiments with antimycin only in Fig S3J and 2H.Experiments should also be conducted in other experimental conditions.As reported above, authors should perform experiments by decreasing ROS species.

Minor points:
It is kindly suggested to perform experiments detecting another important mtROS, such as superoxide anion; it is recommended to perform all experiments in at least another breast cancer cell line.4A is performed only on MDA-MB-231 cells, while for Figure 4 B-C-D-F, there is no evidence about cell lines.Experiments on 4F are performed on B16-F10 without a justification.It would be better to use a more specific inhibitor for Src kinase and not PP2, which specifically inhibits Lck, Fyn, and Hck.The authors can be used Dasatinib; 4J concerning the quantification of cell number in the low chamber using the Boyden transwell assay, the sample treated with PP2 showed far too low levels of migration compared to the Src KD condition.However, it would be better to demonstrate the inhibition of Src activity with a simple WB of the active form of Src (SrcY419).Have authors performed invasion experiments without serum in the culture media?This is not specified in the methods.Usually, invasion should be performed without serum to exclude the effects of cellular proliferation.Furthermore, DMEM and serum have antioxidant properties.It is of great interest to have found an important molecular mechanism regulating the ROS microenvironment.It should be useful to verify whether this mechanism can also regulate another main hallmark for cancer and metastasis, which is the regulation of cell death programs.

Referee #2:
Ren et al. report an interesting relationship between tumor ROS, SRC and cell migration in their manuscript titled "Loss of Myo19 increases metastasis by enhancing microenvironmental ROS gradient and chemotaxis".While this manuscript contains convincing experiments, I pose a number of questions and comments to the authors: In figure 1, it would be more convincing to determine if 4t1 tumors metastasized to the lung.Outgrowth and metastasis are not necessarily the same thing.Being that "metastasis" is the title, experimental manipulation of Myo19 would need to be shown to affect metastasis.How was the "relative H2O2 level" quantified in s3F, similar to 4b (S0038, Beyotime)?Have you considered mitoPy1 (https://www.tocris.com/products/mitopy1_4428)?Is Src the only kinase sensitive to H2O2?Why does Myo19 downregulation increase ROS? Do other perturbations to actin homeostasis increase H2O2-induced chemotaxis?If this migration is actin independent, is this amoeboid migration?Referee #3: In this study by Wu and colleagues, the authors propose three major findings indicated by their experiments: (i) the identification of a ROS gradient in tumor cell spheroids which projects into a H2O2 gradient in the tumor microenvironment; the induction by the H2O2 gradient of tumor cells chemotaxis by activation of Src kinase followed by RhoA GTPase inhibition; the absence of mitochondria cristae remodeling proteins (including actin motor Myosin 19) enhances ROS gradients and promotes spheroid invasion.
The data are clearly presented, but a number of questions are raised by the interpretation, and sometime overinterpretation of the results, which should be carefully addressed.
Is the difference reported in Fig. 1B statistically significant?If yes, statistical analysis employed to show significance should be described.If not, this should be clearly mentioned in the text.The term "propensity" is inappropriate and misleading.Statistical analysis/significance is also missing for data presented in Fig. 1F, making description confusing and interpretation of the results unclear.
At page 5, authors write: "Together, we postulated that loss of cristae remodeling proteins might enhance ROS gradient in tumor spheroid periphery by increasing mitochondria ROS production."It would be a strong support to this hypothesis to show that mitochondria isolated from either control of Myo19 KD cells do indeed show differences in ROS production.
At page 6 it is stated: "Collectively, these results demonstrated the existence of a peripheral ROS gradient in 3D tumor spheroids that could project into a microenvironment H2O2 gradient and can be enhanced by loss of cristae integrity."Again, this overinterpretation of the results shown would be strongly supported by a demonstration of the loss of cristae integrity/decrease in modified cells by comparative high-resolution microscopy or EM.Page 6: "We found that 3D invasion was enhanced in Myo19 KO while decreased in Nrf2 KD spheroids (Supplementary Fig. 6B), and the addition of catalase could inhibit the invasiveness in both WT and Myo19 KO spheroids (Fig. 3C)."For Figure S6, the presentation of representative images for the different experimental conditions should be shown.
End of page 6, cell-autonomous versus non-autonomous effects.The experiments shown in Fig. 3D-G are not convincing.The authors should use a simpler Matrigel-transwell invasion assay to (possibly) exclude cell-autonomous effects on 3D migration.Page 7: "Consistently, WT+KO spheroids displayed enhanced ROS gradient and outspread faster (Fig. 3J and 3K)."An important control is missing: could this increase be reverted by abolishing the ROS gradient in this assay?Immunochemical data in Fig. 4H and 4I: statistically significant differences from more experiments should be presented.Overall, the development of this part is weak and incomplete as it stands.

Referee #1:
This work represents the natural continuation of a recent investigation published by authors in Nature Communication (Mechanical instability generated by Myo19 contributes to mitochondria cristae architecture and OXPHOS" and PMID: 35562374), in which they describe the relationship between Myo19 and mitochondrial metabolism.Here, the authors want to demonstrate that these effects mediated by Myo19 control tumor invasion and metastasis, particularly by regulating the tumor microenvironmental ROS.This new study is fascinating and may extend the knowledge of the important role of ROS microenvironment in tumor growth and, especially, in metastasis.
We thank the reviewer for acknowledging our work.We have made extensive efforts in including experiments in our updated manuscript according to reviewer's insightful suggestions.We feel that this revised manuscript is much improved.However, I would ask the authors to perform some experiments to strengthen their data.One concern is regarding the in vivo experiments reported in Figure 1.The authors injected mice with 4T1 scramble, Mic60 KD, and Myo19 KD cells.As reported in the text, they affirmed that myo19 KO led to higher invasiveness (Fig 1E).However, in the graph, it is the Mic60 KD that gives the higher rate of invasiveness.Myo19KD has a low significance of p=0.0405.This also occurs in Fig. 1F.Have the authors inverted the graphs?
We thank the reviewer for the questions.We did not invert the graphs in Fig. 1F, and Mic60 knockdown (KD) indeed led to higher invasiveness than Myo19 KD, and they both exhibited higher invasion than the control group.One reason to explain this would be that Mic60 KD may lead to much severer damage on mitochondria and influence mitochondria ROS production to a larger extent.It has been reported that Mic60 KD let to very severe cristae disruption, such as onion-shaped cristae [1] ,while Myo19 KO mitochondria also showed lower cristae frequency and displayed onion-shaped cristae only occasionally [2] (Fig. R1.1A).In fact, ROS gradient was more significantly enhanced in Mic60 KD rather than Myo19 KO spheroids (Fig. R1.1B and 1C).These may explain the difference in invasiveness of Mic60 KD and Myo19 KD tumors but would require future investigations.We have also included these results in our revised manuscript (Fig. EV1H).

26th Oct 2023 1st Authors' Response to Reviewers
If the label is correct, authors should explain the choice to focus their investigation on myo19.Throughout the text, the authors tried to justify this only at line 118, but this is a weak connection that further experiments should validate.Furthermore, why did the authors not investigate the contribution of Mic60 throughout the manuscript?If Mic60 is important for ROS production and mitochondrial modeling, why did the authors not measure its levels in human samples?
We thank the reviewer for these questions.As the reviewer stated, this work represents the natural continuation of our recent investigation.Our lab has long been interested in cytoskeleton.Myo19 is so far the only myosin that is reported to primarily localize on mitochondria [3] , but its pathological significance is poorly understood.The comparison between Mic60 and Myo19 KD further supported that the cellular and spheroid phenotypes by Myo19 KD was associated with its role in cristae formation.However, we agreed with the reviewer that the impact of Mic60 in ROS production and tumor progression is of great interest for future studies.During revision, we performed immunohistochemical staining of Mic60 in breast carcinoma patient samples to broaden our conclusion (Fig. R1.2A).We found that lower Mic60 expression was frequently observed in both Metastasis and Non metastasis groups (Fig. R1.2B).In addition, no significant difference in lymphatic metastasis was detected between Mic60 low expression group (L) and moderate expression group (M) (Fig. R1.2C), arguing that Mic60 expression in human breast carcinoma may not be associated with their metastatic status.We further employed the fat mammary pad injection model, and monitored lung metastases of 4T1 breast carcinoma (Fig. R1.2D).Mic60 KD increased the number of lung metastatic foci (Fig. R1.2E and 2F).Together, these results suggested that Mic60 depletion may promote the metastasis of breast carcinoma in mice but not humans.

Figure for referees not shown.
Another thing that is not clear is that even if they found significant increases in the invasiveness, the authors did not find variations in the tumor growth.This is very strange, and the authors should justify this.
We thank the reviewer for pointing this out.Previous studies indicated that tumor invasion is not necessarily accompanied by significantly enhanced tumor growth [4] .Invasion can occur with a small tumor cell number, while large tumors that do not show invasion can also be detected [5] .Here, the tumor cells may not necessarily proliferate more before invading surrounding tissues.Thus, we were able to detect the difference in tumor invasiveness without observing the difference in tumor growth.This phenomenon also suggest that the invasion observed is due to altered cell migration behavior such as chemotaxis, rather than cell growth change.
To be sure about all these in vivo results, I encourage authors to perform a metastatic mouse model in which the primary tumor is removed.
Moreover, can the metastatic potential reduce if mice are injected with cells overexpressing myo19?

Minor points:
It is kindly suggested to perform experiments detecting another important mtROS, such as; We thank the reviewer for the suggestions on detecting another mitochondria ROS.During revision, we investigated the level of mitochondria superoxide anion in WT and Myo19 KO cells using MitoSOX probe.We found that the relative MitoSOX intensity was upregulated in Myo19 KO cells (Fig. R1.5), suggesting that Myo19 depletion upregulated mitochondria superoxide anion level.We have also included this result in our current manuscript (Figure EV2G). it is recommended to perform all experiments in at least another breast cancer cell line.
We thank the reviewer for the suggestions on repeating our experiments in another breast cancer cell line.The existence of ROS gradient in tumor spheroids composed of other tumor cell lines including HeLa, B16-F10 and MCF7, has been confirmed in our last manuscript (Fig. 2D and 2E).In addition to this, we now found that spheroids of 4T1 mouse breast carcinoma cells also displayed the ROS gradient (Fig. R1.6A and 6B).Myo19 KD could also upregulate the cellular ROS level (Fig. R1.6C) and mitochondria

Figure for referees not shown.
ROS level (Fig. R1.6D) in 4T1 cells.Moreover, the invasion of 4T1 spheroids could also be expedited by Myo19 KD (Fig. R1.6E), while the cell-autonomous factors such as migratory ability was not affected, as measured by transwell assay (Fig. R1.6F).Last but not least, we investigated the chemotactic behavior in 4T1 and MCF7 cells, and found extracellular H 2 O 2 gradient could also induce chemotaxis in these cell lines (Fig. R1.6G). 8

Figure for referees not shown.
4A is performed only on MDA-MB-231 cells, while for Figure 4 B-C-D-F, there is no evidence about cell lines.
We apologized for missing this information in the legends.Figure 4A-4D were performed using MDA-MB-231 cells, and Figure 4E-4F using B16-F10 cells.We have also included this information in our updated manuscript.
Experiments on 4F are performed on B16-F10 without a justification.
We thank the reviewer for pointing out this.We employed the imaging based ibidi chemotactic chamber (Fig. 4E-4F) to exclude the cell proliferation artifacts in transwell assays.Since MDA-MB-231 cells harbored higher proliferation rate, and that the proliferating cells were not analyzed due to their halted migration before division and transiently peaked velocity right after cytokinesis, we used the B16-F10 cells instead.B16-F10 cells showed high migration speed and metastatic potential [8]   , which were suitable for this device and subsequent tracking and analysis.We have also added these explanations in the revised manuscript (Page 8, Line 205-209).
It would be better to use a more specific inhibitor for Src kinase and not PP2, which specifically inhibits Lck, Fyn, and Hck.The authors can used Dasatinib; We thank the reviewer for this suggestion.During revision, we evaluated the H 2 O 2 -induced chemotaxis in cells treated with Dasatinib using transwell assay.
Consistent with Src knock down and PP2 treatment, Dasatinib also inhibited cell migration towards the lower chamber upon H 2 O 2 gradient, indicative of inhibited H 2 O 2induced chemotaxis (Fig. R1.7)We have also included this result in our updated manuscript (Fig. 4L).
4J concerning the quantification of cell number in the low chamber using the Boyden transwell assay, the sample treated with PP2 showed far too low levels of migration compared to the Src KD condition.However, it would be better to demonstrate the inhibition of Src activity with a simple WB of the active form of Src (SrcY419).
We thank the reviewer for the suggestions.We took the advice and also included Dasatinib, the more specific Src inhibitor that the reviewer suggested.We assayed the level of phosphorylated Src Y419 using Western blot, and found downregulated active Src in cells treated with PP2 or Dasatinib (Fig. R1.8), suggesting that PP2 and Dasatinib treatments in the transwell assay indeed inhibited Src activity.We have also included these results in the revised manuscript (Figure EV5F).
Have authors performed invasion experiments without serum in the culture media?This is not specified in the methods.Usually, invasion should be performed without serum to exclude the effects of cellular proliferation.Furthermore, DMEM and serum have antioxidant properties.
We thank reviewer for this suggestion.We assayed the 3D invasion of WT and Myo19 KO spheroids in serum-free culture media, and still found higher invasion rate in spheroids   It is of great interest to have found an important molecular mechanism regulating the ROS microenvironment.It should be useful to verify whether this mechanism can also regulate another main hallmark for cancer and metastasis, which is the regulation of cell death programs.
We thank the reviewer for acknowledging our work.ROS, which are considered as double-edge swords, are involved in tumor initiation, proliferation, invasion and angiogenesis [9] .But the excessive ROS would also trigger senescence and multiple cell death pathways [10][11][12] .
The inner core of tumor spheroids and solid tumors are characterized of various cell death pathways, namely apoptosis, necrosis and ferroptosis [13][14][15][16] .Treatment of antioxidants reduced the cell death in the inner spheroids [14,16] , suggesting that cell death in spheroids is associated with their redox environment.It is intriguing to investigate how microenvironmental ROS regulated the cell death pathways in tumor spheroids, and whether mitochondria ROS was involved in this regulation.We have added a section of discussion on this topic (Page 9-10, Line 244-250).We thank the reviewer for acknowledging our work.We have included supplemental experiments to broaden our conclusion according to reviewer's insightful comments and suggestions.We feel that this revised manuscript is much improved.
In figure 1, it would be more convincing to determine if 4t1 tumors metastasized to the lung.Outgrowth and metastasis are not necessarily the same thing.Being that "metastasis" is the title, experimental manipulation of Myo19 would need to be shown to affect metastasis.
We thank the reviewer for this suggestion.During revision, we employed the fat mammary pad injection model, and monitored lung metastasis of 4T1 breast carcinoma (Fig. R2.1A).Consistent with increased invasiveness, we found increased metastatic foci in lungs of Myo19 knock down (Myo19 KD) and Mic60 knock down (Mic60 KD) tumors (Fig. R2.1B and 1C), arguing that loss of Myo19 and Mic60 could promote tumor metastasis.We have also included these results in our current manuscript (Fig. 1G and 1H).

Figure for referees not shown.
How was the "relative H 2 O 2 level" quantified in s3F, similar to 4b (S0038, Beyotime)?
We thank the reviewer for pointing this out."H 2 O 2 level detection kit" (S0038, Beyotime) can be used to detect H 2 O 2 level in cell lysis and culture media.In this assay, H 2 O 2 in the samples could oxidize Fe 2+ to Fe 3+ in the detection reagent, which further reacted with the xylenol orange dye to yield product with maximum absorbance at 560 nm.In Fig. S3F, we inspected the H 2 O 2 level in WT and Myo19 knock out (Myo19 KO) cell lysis, and in Fig. 4B we measured H 2 O 2 level of culture media from upper and lower chambers in transwell assay.We calculated the "relative H 2 O 2 level" to normalize the experimental values.To do this, we first calculated the average H 2 O 2 level of control group (WT group in Fig. S3F and the upper chamber group in Fig. 4B), and then divided the experimental values in each group with this average value.We have also included the analysis in our updated methods session (Page 17, Line 435-437).
We thank the reviewer for this suggestion.During revision, we employed the mitoPY1 to measure H 2 O 2 level in WT and Myo19 KO cells.We found increased mitoPY1 intensity in Myo19 KO cells (Fig. R2.2), indicating upregulated H 2 O 2 level.We have also included this result in our revised manuscript (Figure EV2J).We thank the reviewer for this question.Some other protein tyrosine kinases have also been reported sensitive to redox stimulation, such as the Src family proteins Fyn and Lyn.We focused on Src as a candidate because it is not only sensitive to H 2 O 2 stimulation through direct cysteine oxidation [17] , but also well-known to regulate cell adhesion and migration [18] .Notably, the oxidatively activated Src has been reported to promote cell migration [19,20] and the role of Src in chemotaxis has also been demonstrated [21] .These prompted us to study Src.We would like to further screen for H 2 O 2 chemotactic "sensors" in our future work.

Why does Myo19 downregulation increase ROS?
We thank the reviewer for pointing this out.Mitochondria are one of the major sources for cellular ROS production, and mitochondria ROS are produced during OXPHOS [22] , which takes place at cristae.Absence of cristae sculpturing proteins such as Myo19 and Mic60 disrupts cristae, which enhances electron leakage and promotes ROS production [1,23] .We apologize for missing this rationale in the previous manuscript, and we have added this in the revised manuscript (Page 4, Line 98-99).During revision, we have also examined mitochondria fine structure with electron microscopy (Fig. R2.3) and verified that loss of Myo19 or Mic60 disrupted cristae structure, which may then increase mitochondria ROS.We have also included the electron microscopy data in our revised manuscript (Figure EV1H).

Do other perturbations to actin homeostasis increase H 2 O 2 -induced chemotaxis? If this migration is actin independent, is this amoeboid migration?
We thank the reviewer for the suggestions.The role of actin in directed migration has been extensively studied [24,25] .We employed transwell assay and investigated the chemotaxis of cells treated with Latrunculin B (LatB), which inhibits actin polymerization.Low concentration of LatB did not completely abolished cell migration towards the lower chamber but partially inhibited the chemotactic efficiency (Fig. R2.4A).However, global disruption of the actin cytoskeleton by LatB may affect chemotaxis through inhibiting intrinsic migration machineries other than altering the H 2 O 2 chemotaxis.
Actin plays an important role in both mesenchymal and ameboid migration.To evaluate whether H 2 O 2 -induced chemotaxis was mesenchymal or ameboid migration, we assayed the 2D migration of cells within the H 2 O 2 gradient.We found the migrating cells adopted a well-spread morphology and displayed strong adhesion with the substrate (Fig. R2.4B), which is one of the characteristics of mesenchymal migration [26] .This result indicated that in our experimental condition, H 2 O 2 -induced chemotaxis is likely to be mesenchymal migration.

Referee #3:
In this study by Wu and colleagues, the authors propose three major findings indicated by their experiments: (i) the identification of a ROS gradient in tumor cell spheroids which projects into a H2O2 gradient in the tumor microenvironment; the induction by the H2O2 gradient of tumor cells chemotaxis by activation of Src kinase followed by RhoA GTPase inhibition; the absence of mitochondria cristae remodeling proteins (including actin motor Myosin 19) enhances ROS gradients and promotes spheroid invasion.The data are clearly presented, but a number of questions are raised by the interpretation, and sometime overinterpretation of the results, which should be carefully addressed.
We thank the reviewer for acknowledging our work.We have included supplemental experiments and modified the text to avoid misleading statements in our updated manuscript accordingly.We have also modified the manuscript to exclude overinterpretation of the results.We feel that this revised manuscript is much improved.
Is the difference reported in Fig. 1B statistically significant?If yes, statistical analysis employed to show significance should be described.If not, this should be clearly mentioned in the text.The term "propensity" is inappropriate and misleading.
We apologize for the inappropriate and misleading descriptions.The statistical analysis of Fig. 1B was conducted and shown in Fig. 1C.To avoid misunderstandings, we have deleted the word "propensity" and more specifically interpreted this result in the revised manuscript (Page 3, Line 71).Fig. 1B displayed the probability distribution of Myo19 expression in the two groups, and revealed a left shift of the peak value in the Metastasis group, which indicated a higher metastatic frequency in the lower Myo19 expression samples.Next, we sought to divide the samples into Myo19 Low, Moderate and High groups, and found higher frequency of metastatic incidents in the Low group compared to the Moderate group (Fig. 1C).Together, these results suggested that lower-expressed Myo19 in tumors was associated with higher metastasis.
Statistical analysis/significance is also missing for data presented in Fig. 1F, making description confusing and interpretation of the results unclear.
We apologize for the confusing descriptions.Loss of Myo19 promoted the local invasion of mouse breast carcinoma into normal skeletal muscles, and loss of Mic60 promoted the invasion into skeletal muscle and skin tissues.To more clearly displayed these results and avoid misinterpretations, we replaced the graph in Fig. 1F with a table to elucidate the sites where the tumor cells were invading, and added the statistical significance below the number (Fig. 1F).At page 5, authors write: "Together, we postulated that loss of cristae remodeling proteins might enhance ROS gradient in tumor spheroid periphery by increasing mitochondria ROS production."It would be a strong support to this hypothesis to show that mitochondria isolated from either control of Myo19 KD cells do indeed show differences in ROS production.

Percentage
We thank the reviewer for the suggestions.During revision, we isolated mitochondria in WT and Myo19 KO cells (Fig. R3.1A), and assayed the H 2 O 2 level in the lysis of isolated mitochondria as the reviewer suggested.We found that Myo19 KO mitochondria displayed higher H 2 O 2 level (Fig. R3.1B).In addition, we investigated the level of mitochondria superoxide anion in WT and Myo19 KO cells using MitoSOX.We found that the MitoSOX intensity was upregulated in Myo19 KO cells (Fig. R3.1C).Together, these results strengthened the notion that Myo19 KO induced mitochondria ROS production.
We have also included these results in our revised manuscript (Figure EV2E-2G).
At page 6 it is stated: "Collectively, these results demonstrated the existence of a peripheral ROS gradient in 3D tumor spheroids that could project into a microenvironment H 2 O 2 gradient and can be enhanced by loss of cristae integrity."Again, this

Figure for referees not shown.
overinterpretation of the results shown would be strongly supported by a demonstration of the loss of cristae integrity/decrease in modified cells by comparative high-resolution microscopy or EM.
We apologize for this overinterpretation and thank the reviewer for the suggestions.It has been reported by us and others that absence of Myo19 or Mic60 could disrupt cristae integrity, as indicated by electron microscopy [1,2,27] and Hessian-SIM super resolution microscopy [28] .To further strengthen this conclusion, we re-inspected the crista morphology in WT, Myo19 KO and Mic60 KD cells using electron microscopy in our current experimental conditions as the reviewer suggested.We found that absence of Myo19 or Mic60 could affect cristae morphology (Fig R3 .2).We have also included this result in our revised manuscript (Figure EV1H).
Page 6: "We found that 3D invasion was enhanced in Myo19 KO while decreased in Nrf2 KD spheroids (Supplementary Fig. 6B), and the addition of catalase could inhibit the invasiveness in both WT and Myo19 KO spheroids (Fig. 3C)."For Figure S6, the presentation of representative images for the different experimental conditions should be shown.
We apologize for missing these representative images and thank the reviewer for the suggestions.We have added the representative images of the 3D invasion of WT/Myo19 We thank the reviewer for the suggestions.During revision, we assayed WT and Myo19 KO cell invasion using Matrigel-coated transwells and found that the migratory cells to the lower chamber remained steady upon Myo19 depletion (Fig. R3.4), which argued against cell-autonomous effects on 3D migration.Figure for referees not shown.outspread faster (Fig. 3J and 3K)."An important control is missing: could this increase be reverted by abolishing the ROS gradient in this assay?
We apologized for the ambiguous descriptions.In this assay, we did perform the control group with spheroids of abolished ROS gradient, which was the KO+WT group (with Myo19 KO preformed core and WT layered around the periphery).Reconstructed spheroids of KO+WT group harbored decreased ROS gradient since Myo19 KO cells displayed higher ROS level and WT cells displayed lower ROS.Indeed, compared to WT+WT spheroids with normal ROS gradient and invasion rate, KO+WT spheroids displayed decreased ROS gradient and slowed spreading (Fig. 2J and 2K).We have rewritten this part with more specific descriptions in the revised manuscript (Page 7, Line 182-185).
Immunochemical data in Fig. 4H and 4I: statistically significant differences from more experiments should be presented.Overall, the development of this part is weak and incomplete as it stands.
We apologize for missing this information and thank the reviewer for the suggestions.We have included the Western blot in Fig. 4H and 4I with three independent experiments as the reviewer suggested, and found that H 2 O 2 stimulation upregulated phosphorylated Y419 Src level (Fig. R3.5A) and downregulated active RhoA level (Fig. R3.5B) using Student's t-test.These results strengthened our findings that H 2 O 2 could activate Src and inhibit RhoA.Considering that Src inhibition and RhoA activation could both inhibit H 2 O 2 -induced chemotaxis (Fig. 4J), these results suggested that H 2 O 2 could induce chemotaxis through Src-RhoA signaling.We have also included these results in our revised manuscript (Fig. 4H-4K).Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
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